EP3530652A1 - Modifiziertes phenylphthalimid und pharmazeutische zusammensetzung damit als wirkstoff - Google Patents

Modifiziertes phenylphthalimid und pharmazeutische zusammensetzung damit als wirkstoff Download PDF

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EP3530652A1
EP3530652A1 EP17862847.5A EP17862847A EP3530652A1 EP 3530652 A1 EP3530652 A1 EP 3530652A1 EP 17862847 A EP17862847 A EP 17862847A EP 3530652 A1 EP3530652 A1 EP 3530652A1
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peg11
group
cells
chloroform
mmol
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French (fr)
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EP3530652A4 (de
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Yutaka Hattori
Maiko MATSUSHITA
Daiju ICHIKAWA
Shuji Aida
Takeshi Sugai
Taketo Yamada
Hiroshi Yanagawa
Yoko Ogawa
Noriko Tabata
Yuko YONEMURA
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Idac Theranostics Inc
Keio University
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Idac Theranostics Inc
Keio University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the present invention relates to a novel phenylphthalimide derivative and a pharmaceutical composition containing the same as an active ingredient.
  • the present invention more specifically relates to a phenylphthalimide derivative prepared by modification of phenylphthalimide with polyethylene glycol (PEG) or a hydroxy-alkoxy group, a method for producing the same, and a pharmaceutical composition such as an anticancer agent comprising the phenylphthalimide derivative.
  • MM Multiple myeloma
  • B-cells plasma cells
  • MM causes the presentation of various characteristic symptoms including osteolytic lesion, hematopoietic injury (particularly, anemia), hypercalcemia, renal dysfunction, immunodeficiency, and extramedullary plasmacytoma, etc.
  • the number of national patients with MM is estimated to be 14,000, approximately 4,000 new MM patients are diagnosed annually, and annual death number is 4,066.
  • MM is common in elderly people. Our country will be a super-aging society and thus the number of MM patients will steadily increase. Worldwide, the number of MM patients reaches as high as 750,000.
  • Non-patent document 1 Non-patent document 2
  • Non-patent document 3 Protein cereblon (cereblon; CRBN) that binds as teratogen to thalidomide has been recently identified. Furthermore, compounds analogous to thalidomide (lenalidomide, pomalidomide) referred to as immunomodulatory drugs (IMiDs) have emerged and been used for countermeasures against MM, a refractory disease, (Non-patent document 4; Non-patent document 5; Non-patent document 6). However, all of these cases will be resistant to these novel drugs within several years, and most cases will be fatal.
  • IiDs immunomodulatory drugs
  • drugs with other mechanisms include drugs, the efficacy of which is confirmed by the use thereof as a single drug (Non-patent document 12) or the same in combination with lenalidomide.
  • an anti-CD38 antibody (daratumumab) and an anti-PD-Ll antibody (atezolizumab) have been administered in combination.
  • antibody medicines are expensive and some of these medicines are injections and are unable to be applied via oral medication, and thus cannot always be optimum in view of patients' convenience.
  • a novel compound effective for even a case with high-risk chromosomal abnormality should be searched for, and the prognosis should be improved.
  • the present inventors have synthesized to date many phthalimide derivatives, and discovered a compound TC11 (2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione; Fig. 1 ) having a high drug effect using as an index the inhibition of the proliferation of many myeloma cell line panels including high-risk myeloma cell lines that the present inventors had established themselves (Non-patent document 13; Patent document 1).
  • TC11 comprises a phthalimide ring and a diisopropylphenyl group as the backbone, having an amino group at position 5 of the phthalimide ring (Non-patent document 14; Non-patent document 15; Patent document 2).
  • TC11 lacks a glutarimide ring that is a CRBN binding site of thalidomide, lenalidomide or the like (Non-patent document 6).
  • Nucleophosmin 1 (NPM1/B23.1) and ⁇ -tubulin being located as a target protein of TC11 in centrosomes during mitotic phase and having a function of controlling centrosome duplication have been identified (Non-patent document 11).
  • Non-patent document 11 Similar phenomena of apoptosis induction have also been observed in experiments of the knockdown of NPM1 by siRNA using HeLa cells. Furthermore, the present inventors have also revealed that TC11 is effective against MM resistant to lenalidomide, and thalidomide in a human MM xenograft model established using immunodeficient mice (Non-patent document 16).
  • PEG is a nontoxic and nonimmunogenic polymer, and has a feature such that the size or the side-chain structure can be varied depending on its application.
  • PEGylation of target proteins or peptides has been focused and used as a method for improving the pharmacokinetic properties and the immunologic properties of proteins and peptides having poor stability in vivo or for targeting etc., with the use of the above features (Non-patent document 17).
  • An object of the present invention is to provide a novel phenylphthalimide derivative. Another object of the present invention is to provide a pharmaceutical composition comprising the novel derivative. Another object of the present invention is to provide a therapeutic agent for various cancers including MM and immune abnormality, comprising the novel derivative.
  • TC11 has been problematic in its low water solubility in view of development thereof as a drug. It has been observed that in a tumor-transplanted mouse model, most TC11 administered via intraperitoneal injection remains unabsorbed within the abdominal cavity. Hence, TC11 should have improved water solubility upon its formulation.
  • salt formation of amino groups bound to a phthalimide ring with hydrochloric acid and maleic acid was attempted, but almost no salt formation of TC11 was confirmed. Therefore, through modification with PEG, hydroxyalkoxy, sugars, and carbohydrate, etc., the present inventors aimed at improving the water solubility, blood kinetics and drug effect of TC 11.
  • the present inventors discovered a phenylphthalimide derivative, which can be easily synthesized, and has significantly improved water solubility and blood kinetics, low toxicity, no side effect such as teratogenicity, and a high drug effect against various cancers including MM with high-risk chromosomal abnormality, and immune abnormality, and thus completed the present invention.
  • R 1 is a group containing PEG or a hydroxy C 1-5 alkoxy group binding to position 4 or 5
  • R 2 is an amino group at position 6
  • R 3 and R 4 are each independently a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, or an aryloxy group.
  • R 1 is preferably a group containing PEG, and is further preferably a group in which PEG binds to a phthalimide ring via an ester bond.
  • R 1 may also be a hydroxy C 1-5 alkoxy group.
  • R 1 preferably binds to position 5.
  • R 3 and R 4 are both isopropyl groups preferably located at position 2' and position 6'.
  • the present invention provides a pharmaceutical composition comprising the above derivative.
  • the pharmaceutical composition is preferably an anticancer agent.
  • the anticancer agent is preferably used for multiple myeloma.
  • the derivative of the present invention can be easily synthesized, and has significantly improved water solubility and blood kinetics, low toxicity, no side effect such as teratogenicity, and a high drug effect on various cancers including MM with high-risk chromosomal abnormality and immune abnormality, and thus is advantageous as a pharmaceutical product.
  • TC11 is known to have an anticancer effect and a high drug effect particularly on myeloma (Non-patent document 13).
  • Nucleophosmin 1 (NPM1/B23.1) and ⁇ -tubulin being located in centrosomes during mitotic phase as a target protein of TC11 and having a function of controlling centrosome duplication have been identified (Non-patent document 13). It has been revealed that when tumor cells are treated with TC11, TC11 inhibits centrosomal clustering, as a result, induces multi-polarization and further multinucleation of mitotic cells, thereby inducing apoptosis (Non-patent document 13).
  • TC11 inhibits oligomerization of NPM1.
  • NPM1 is considered to exhibit cell biological functions via oligomerization, while TC11 is considered to suppress the functions.
  • TC11 inhibits ⁇ -tubulin polymerization. It is thus inferred that mitotic catastrophe is induced by such inhibition.
  • TC11 induces apoptosis of myeloma cells with various mechanisms including (1) inhibition of NPM1 oligomerization, (2) inhibition of ⁇ -tubulin polymerization, followed by the suppression of TC11-induced apoptosis by CGP74514A, and (3) induction of cell death by excessive activation of CDK1, etc.
  • the present inventors synthesized various derivatives resulting from modification of TC11 with polyethylene glycol (PEG), hydroxyalkoxy, sugars such as glucose, carbohydrate, and the like, and intensively searched for activity of suppressing the growth of various cancers including MM. Specifically, the present inventors examined a phenylphthalimide derivative represented by the following general formula.
  • R 1 contains a PEG group binding to any one of position 4, 5, 6, and 7 of a phthalimide ring via an ester group or an ether group
  • R 1 contains a hydroxy C 1-5 alkoxy group binding to any one of position 4, 5, 6, and 7 of the phthalimide ring
  • R 1 contains a monosaccharide, a derivative of a monosaccharide, a disaccharide, a polysaccharide, or a sugar alcohol binding to any one of position 4, 5, 6, and 7 of the phthalimide ring.
  • R 2 represents an amino group, a hydroxy group, a nitro group, an O-alkyl group, or an S-alkyl group located at position 6 or 5 of the phthalimide ring.
  • R 3 and R 4 may be the same or different, and each represents a hydrogen atom, an alkyl group, a haloalkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylamino group, an alkylthio group, an aralkyl group, or an aryloxy group.
  • R 3 and R 4 are each independently a hydrogen atom, an alkyl group or an aryl group
  • R 1 is a group containing PEG or a hydroxy C 1-5 alkoxy group binding to position 4 or 5
  • R 2 is an amino group at position 6
  • R 3 and R 4 are each independently a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, or an aryloxy group.
  • R 1 may be a group containing PEG.
  • PEG generally refers to PEG represented by a structural formula CH 3 O(CH 2 CH 2 O) n H wherein n is between 5 and 15 (preferably 6 and 15, further preferably 8 and 13).
  • PEG may directly bind to a phthalimide ring via terminal oxygen atom (binding via an ether bond), or to a phthalimide ring via a divalent organic group.
  • any organic group can be used as long as it does not inhibit the anticancer activity of the derivative, and in general, polyoxyalkylene in which a non-terminal oxygen atom is substituted with an ester group (binding via an ester bond) can be used.
  • Alkylene has a carbon number of generally 2 to 4, and a polymerization degree of generally 2 to 3.
  • polyoxyethylene an example thereof is a -CH 2 -CH 2 -C(O)-O-CH 2 -CH 2 -O- structure.
  • An end of PEG, which is on the side not binding to a phthalimide ring may be OH or an alkyl group having a carbon number of 1-3, and is generally a methyl group.
  • R 1 is preferably a group in which PEG binds to a phthalimide ring via polyoxyalkylene wherein a non-terminal oxygen atom is substituted with an ester group, specifically, an ester bond.
  • Hydroxyalkoxy-TC11 is a compound generated by hydrolysis of PEG 11 (E)-TC11 by in vivo esterase. Accordingly, in the case of binding via an ester bond, more desirable pharmacokinetics can be exhibited as a result of in vivo hydrolysis.
  • a hydroxyalkoxy derivative also exhibits an anticancer effect, and thus R 1 may also be a hydroxy C 1-5 alkoxy group.
  • a hydroxy-alkoxy group has a carbon number of preferably 1 to 3, and further preferably 2.
  • R 1 examples include CH 3 O(CH 2 CH 2 O) n CH 2 CH 2 O-, and CH 3 O(CH 2 CH 2 O) n CH 2 CH 2 C(O)OCH 2 CH 2 O- wherein "n" is as described above.
  • R 1 preferably binds to position 5.
  • R 2 is an amino group binding to position 6. It is considered that an amino group should be present at position 6 of a phthalimide ring for the derivative to exhibit anticancer activity.
  • R 1 group is bound to position 5 or position 4 in order to avoid the inhibition of the effect of the amino group.
  • phthalimide rings are symmetric, and thus the relationship between position 6 and position 5 or position 4 is equivalent to that between position 5 and position 6 or position 7.
  • R 3 and R 4 are each independently a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, or an aryloxy group.
  • An alkyl group is generally an alkyl group having a carbon number of 1 to 6 (preferably 1 to 3). Examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and a t-butyl group.
  • An aryl group is generally an aryl group having a carbon number of 6 to 10. An example thereof is a phenyl group.
  • Aryl may be substituted with an alkyl group.
  • the alkyl group may be similar to the above alkyl.
  • An aralkyl group is an aralkyl group wherein an alkyl portion has a carbon number of 1 to 6 (preferably 1 to 3).
  • the aryl portion may be similar to the above aryl group. Examples thereof include a benzyl group, and a phenethyl group.
  • Alkyl portions in an alkyl group and an aralkyl group may be linear or branched.
  • An aryloxy group may be a group in which oxygen atoms bind to the above aryl group and aralkyl group.
  • R 3 and R 4 are not particularly limited.
  • R 3 is substituted with a phenyl group at position 2' or position 6', or R 3 is substituted with an oxyphenyl group at position 4'.
  • R 3 and R 4 are both isopropyl groups, and preferably present at position 2' and position 6'.
  • the structure on the phenyl ring side should be hydrophobic for the derivative to exhibit anticancer activity. Accordingly, it is inferred that anticancer activity is exhibited based on the range of the above R 3 and R 4 . Hence, it is also inferred that the structure on the phenyl ring side should not be PEGylated. As described later in Example 14, the structure on the phenyl ring side is also a structure that does not bind to the cereblon protein.
  • the derivative of the present invention can be produced through combination of known synthesis techniques. This will be described with specific examples as follows.
  • PEG11(E)-TC11 ( Fig. 1 ) of the present invention can be produced by a method for producing the TC11 derivative (PEG11(E)-TC11) through PEGylation of TC11 via an ester bond, such as the following method when a reactive group for modification is mPEG 1-COOH.
  • the compound of the present invention PEG11(E)-TC11 prepared via modification with PEG, can be obtained as follows.
  • 4-hydroxyphthalic acid is nitrated with guanidine nitrate to give a mixture of a 5-nitro compound and a 3-nitro compound of 4-hydroxyphthalic acid, and then the mixture is dehydrated with phosphorus pentoxide so as to be able to give a hydroxyphthalic anhydride mixture of a 5-nitro anhydride and a 3-nitro anhydride.
  • the mixture is reacted with 2,6-diisopropylaniline, so as to be able to give a diisopropylphenyl phthalimide derivative (1a and 1b).
  • the mixture is purified by silica gel chromatography, so that 1a and 1b can be separated from each other.
  • 1a is reacted with ethylene bromohydrin, so as to be able to give hydroxyethoxy-la.
  • the hydroxyethoxy-la is esterified with mPEG11-COOH to give PEG11(E)-1a, and then PEG11(E)-1a is subjected to catalytic reduction with a hydrogen gas in the presence of a Pd/C catalyst, so that PEG11(E)-TC11 can be finally obtained.
  • PEG11(0)-TC11 of the present invention can be produced by a method for producing the TC11 derivative (PEG11(O)-TC11) through PEGylation ofTC11 via an ether bond such as the following method when a reactive group for modification is mPEG11.
  • Hydroxyethoxy-1a synthesized in advance is tosylated with para-toluenesulfonyl chloride (TsCl) to synthesize tosylethoxy-1a, mPEG11 is reacted with the resultant to obtain PEG(O)-1a, and then catalytic reduction is finally performed with a hydrogen gas in the presence of a Pd/C catalyst, so that PEG11(O)-TC11 can be obtained.
  • TsCl para-toluenesulfonyl chloride
  • a derivative having another substituent, such as PEG5(E)-TC11 can be similarly produced by adequately substituting a starting material or the like.
  • the present invention provides a pharmaceutical composition comprising the above derivative.
  • the pharmaceutical composition is preferably an anticancer agent.
  • the anticancer agent is preferably used for multiple myeloma.
  • a phenylphthalimide derivative as an active ingredient can be formulated into a preparation (pharmaceutical composition) using a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include an excipient and a base.
  • the preparation may contain a generally employed additive.
  • the dosage form thereof is selected as appropriate depending on the route of administration.
  • the phenylphthalimide derivative may be formulated into preparations such as tablets, capsules, granules, powders, or syrups for peroral administration, or can be formulated into preparations such as injection preparations, or suppositorries for parenteral administration via intraperitoneal, intravenous, or subcutaneous injection.
  • the preparation also include a preparation prepared by packaging the phenylphthalimide derivative as an active ingredient and another anticancer agent separately and then integrating the packages into a single preparation.
  • the anticancer agent of the present invention can be administered to a patient who is receiving or will receive anticancer drug treatment.
  • a phenylphthalimide derivative may be used independently or in combination with another drug (for example, another anticancer agent).
  • the dosage of the phenylphthalimide derivative is selected as appropriate depending on target anticancer drug treatment, patient's conditions, and the like.
  • the phenylphthalimide derivative when the phenylphthalimide derivative is administered to a human, the phenylphthalimide derivative can be administered at a dosage of about 0.1 to 20 mg/kg (body weight) per day, preferably about 0.1 to 0.5 mg/kg (body weight) per day in a single dose or several doses via peroral administration or parenteral administration including intravenous administration and subcutaneous administration.
  • the dosage and the frequency of administration can be varied adequately depending on the symptom, age, and the method of administration, for example.
  • the phenylphthalimide derivative can be used for treatment of cancers including, particularly, hematologic malignancies such as myeloma, malignant lymphoma, leukemia, and myelodysplastic syndrome and solid cancers such as colon cancer, lung cancer, renal cell cancer, mammary cancer, brain tumor, ovarian carcinoma, melanoma, stomach cancer, and prostate cancer.
  • hematologic malignancies such as myeloma, malignant lymphoma, leukemia, and myelodysplastic syndrome
  • solid cancers such as colon cancer, lung cancer, renal cell cancer, mammary cancer, brain tumor, ovarian carcinoma, melanoma, stomach cancer, and prostate cancer.
  • the phenylphthalimide derivative can be advantageously used for multiple myeloma.
  • the reaction solution was poured into ice-cold H 2 O (400 ml), and then the flask was washed with 80 ml of ice-cold H 2 O. The total amount was about 550 ml.
  • the solution was poured into a 1-L separatory funnel, and then extracted twice with ethyl acetate (170 ml ⁇ 2).
  • the resulting ethyl acetate layer was washed twice with a saturated saline solution (70 ml ⁇ 2), dehydrated with anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness.
  • the nitrated product 3.66 g (13.15 mmol, yield 95.8%) was obtained as a pale yellow powder by the above step.
  • the reaction product was found to be an about 1:1 mixture of 5-nitro-4-hydroxyphthalic acid and 3-nitro-4-hydroxyphthalic acid.
  • the product ratio of the 5-nitro compound and the 3-nitro compound was 1:1 as a result of 1 H NMR spectroscopy.
  • a mixture (1.22 g, 5.37 mmol) of 5-nitro-4-hydroxyphthalic acid and 3-nitro-4-hydroxyphthalic acid were added to a 500-ml three-necked flask, and then the mass was pulverized using a glass rod. After 1 hour of drying under vacuum, dehydrated toluene (150 ml) was added. However, the resultant was practically insoluble and thus was subjected to ultrasonication to give a suspension that was homogenous as possible.
  • SICAPENT (14.4 g, as P 2 O 5 : 10.8 g, 76.08 mmol) was weighed in a beaker, and then immediately added into the flask under a nitrogen gas using a funnel and a spatula.
  • SICAPENT is highly hygroscopic, it should be added quickly.
  • the funnel was washed with the remaining dehydrated toluene (33 ml), and then under an argon gas, heated under reflux at 100°C for a whole day and night. After cooling to room temperature, the reaction solution was filtered off, the resultant was washed with toluene, and then the filtrate (with slight clouding) was evaporated to dryness.
  • the reaction product 571.6 mg (2.73 mmol, yield 50.8%) was obtained as a pale yellow powder by the above step.
  • the product ratio of the 5-nitro compound and the 3-nitro compound was 1:0.6 as a result of 1 H NMR spectroscopy.
  • 2,6-Diisopropylaniline (587mg, 3.31mmol) was weighed in a 50-ml two-necked eggplant flask, acetic acid (2 ml) was added, and then the mixture was stirred. A mixture (571.6 mg, 2.73 mmol) of 5-nitro and 3-nitro-4-hydroxyphthalic anhydrides was added to and dissolved in the remaining acetic acid (11.5 ml), followed by heating for a whole day and night under reflux at 140°C. After cooling to room temperature, the reaction solution was poured into 75 ml of H 2 O (in an amount about 5 times that of acetic acid), to immediately give a beige fine precipitate. The solution was gently stirred for 30 minutes, and then the resulting precipitate was filtered off (see note). After washing with 100 ml of 5% acetic acid, the filter paper including the precipitate was dried.
  • a light brown powder (687 mg) was obtained by the above step.
  • the powder adhered to the filter paper was dissolved with ethyl acetate and then evaporated to dryness, so that 273 mg of a similar light brown powder was obtained.
  • the resultant was a mixture (compound 1a and compound 1b) in which 2,6-diisopropylaniline was added to the 5-nitro compound and to the 3-nitro compound, respectively.
  • the purified product was identified as 1a by 1 H-NMR spectroscopy in deuterated chloroform. Moreover, under the above TLC conditions, only a single spot of 1a was confirmed. The yield was 628.8 mg (31.8%).
  • the reaction product was identified as hydroxyethoxy-la as a result of 1 H-NMR spectroscopy in deuterated chloroform.
  • the yield of hydroxyethoxy-1a was 567.2 mg (1.38 mmol, 80.7%).
  • Hydroxyethoxy-la (228.4 mg, 0.554 mmol) was weighed in a 20-ml pear-shaped flask and mPEG11-COOH (401.5 mg, 0.682 mmol) was weighed in a 10 ml pear-shaped flask, followed by about 1 hour of drying under vacuum.
  • Dehydrated DMF (1.6 ml) and triethylamine (257 ⁇ l, 1.85 mmol) were added to a 50-ml two-necked eggplant flask, and then 4-dimethylaminopyridine (6.96 mg, 0.057 mmol) and 2-methyl-6-nitrobenzoic anhydride (235.1 mg, 0.683 mmol) were further added under a nitrogen gas flow.
  • PEG11(E)-1a (365 mg, 0.371 mmol) was added to a 200-ml two-necked eggplant flask, and then dissolved in 43 ml of tetrahydrofuran:ethanol (1:1). After addition of 5% Pd/C (172.5 mg) under an argon gas flow, substitution with a hydrogen gas was performed, and then to stirring at room temperature for a whole day and night. The reaction solution was filtered using sellite, and then the filtrate was evaporated to dryness, so that 402.1 mg of a residue (yellow green viscous liquid) was obtained.
  • PEG5(E)-TC11 was synthesized from the hydroxyethoxy-la by the following method.
  • Hydroxyethoxy-1a (666.6 mg, 1.62 mmol) was weighed in a 100-ml eggplant flask, dried under vacuum for about 1 hour, and then dissolved by the addition of dehydrated toluene (43 ml). While stirring on ice, para-toluene sulfonyl chloride (623.3 mg, 3.27 mmol) was added under a nitrogen gas flow. 4-dimethylaminopyridine (400.2 mg, 3.28 mmol) was subsequently added to cause precipitation, but left to stand until the temperature returned to room temperature, followed by stirring for a whole day and night.
  • the product was identified as tosylethoxy-1a by 1 H-NMR spectroscopy in deuterated chloroform.
  • the yield was 503.7 mg (0.888 mmol, 55%) and the same of the unreacted hydroxyethoxy-la was 144.4 mg (0.35 mmol, 22%).
  • Molecular sieve 3A (1.0 g) was weighed in a small beaker. Twenty five seconds of microwave heating and 20 minutes of cooling under vacuum were repeated 3 times to activate molecular sieve 3A, and then the resultant was added to a 20-ml two-necked eggplant flask under a nitrogen gas flow. mPEG11 (307.6 mg, 0.595 mmol) was weighed in a 20 ml pear-shaped flask, decompressed for 1 hour, dissolved in dehydrated DMF (2 ml), added to the flask of molecular sieve 3A, and then stirred at room temperature for 30 minutes to 1 hour.
  • Sodium hydride (46.4 mg, 27.8 mg as sodium hydride, 1.16 mmol) was weighed in a 10-ml two-necked eggplant flask. Mineral oil adhered thereto was washed 3 times with dehydrated hexane, dried under vacuum, and then suspended in dehydrated DMF (2.9 ml). The previous DMF solution of mPEG11 was stirred on ice for cooling. The suspension of sodium hydride was slowly added dropwise to the solution and then the solution was stirred at room temperature for 1.5 hours.
  • tosylethoxy-1a (157.9 mg, 0.278 mmol) weighed in advance in a 20 ml pear-shaped flask with stirring on ice, dried under vacuum for 2 to 3 hours and then dissolved in dehydrated DMF (1.4 ml) was added dropwise. The resultant was stirred on ice for 7 to 10 minutes. Next, a supernatant of the reaction solution was transferred into a separatory funnel, to which 0.1 M phosphate buffer pH7.0 (5 ml) had been added. The reaction flask and the molecular sieve were washed with ether (12 ml), and then the first extraction was performed.
  • PEG11(O)-1a (165.5 mg, 0.182 mmol) was added to a 200-ml two-necked eggplant flask, and then dissolved in 40 ml of tetrahydrofuran:ethanol (1:1). After addition of 5% Pd/C (135.6 mg) under an argon gas flow, substitution with a hydrogen gas was performed, and then the resultant was stirred at room temperature for a whole day and night. The reaction solution was filtered using sellite, and then the filtrate was evaporated to dryness.
  • Hydroxyethoxy-la (309.1 mg, 0.749 mmol) was weighed in a 50-ml two-necked eggplant flask, dried under vacuum for about 1 hour, and then dissolved in dehydrated acetonitrile (4.5 ml).
  • Diisopropylethylamine 256.6 ⁇ l, 1.50 mmol
  • acetobromo- ⁇ -D-glucose (616.9 mg, 1.50 mmol) weighed in a 20 ml pear-shaped flask and dissolved in dehydrated acetonitrile (5.5 ml) were added, followed by heating under reflux at 80°C for a whole day and night.
  • the reaction solution was transferred into a 50-ml eggplant flask and then evaporated to dryness, thereby obtaining 1.15 g of a residue.
  • the residue was dissolved in 2.5 ml of chloroform, and then injected into the upper part of a silica gel column ( ⁇ 3.5 ⁇ 10.5 cm; 100.8 ml) subjected to swelling in chloroform. After washing with chloroform:ethyl acetate (90:1), a target reaction product was separated with chloroform:ethyl acetate (80:1).
  • Glu(OAc)-1a (739.5 mg, 0.939 mmol) was added to a 300-ml two-necked eggplant flask, and then dissolved in 100 ml of tetrahydrofuran:ethanol (1:1). After addition of 5% Pd/C (460.9 mg) under an argon gas flow, substitution with a hydrogen gas was performed, and then stirring was performed for a whole day and night at room temperature. The reaction solution was filtered using sellite, and then the filtrate was evaporated to dryness.
  • Glu(OAc)-TC11 (679.5 mg, 0.897 mmol) was added to a 100-ml two-necked eggplant flask, and then dissolved in dehydrated methanol (25 ml). Trimethylamine (5.6 ml, 40.2 mmol) was added and then the mixture was stirred for a whole day and night at room temperature. The reaction solution was dissolved in methanol, transferred into a 100-ml eggplant flask, and then evaporated to dryness. Subsequently, toluene was added (not dissolved) and then the resultant was evaporated twice to dryness, thereby removing triethylamine. Furthermore, evaporation to dryness was repeated 3 times with ethyl acetate.
  • the powder was dissolved in 30 ml of chloroform, and then injected into the upper part of a silica gel column ( ⁇ 3.5 cm ⁇ 6 cm; 57.6 ml) subjected to swelling in chloroform. After washing sequentially with chloroform: ethyl acetate (100), (50:1), and then (20:1), and then a target reaction product was eluted with chloroform:ethyl acetate (5:1).
  • PEG11(E)-TC11(A) and PEG11(O)-TC11(B) were examined for stability in mouse blood.
  • PEG11(E)-TC11 and PEG11(0)-TC11 were administered intraperitoneally at 59.1 mg/kg BW and 54.6 mg/kg BW, respectively, in such a manner that each molar concentration was equivalent to that of 20 mg/kg BW of TC11.
  • a tail vein was punctured with a 26G needle for bleeding, and then about 100 ⁇ L of blood was collected using a heparinized hematocrit capillary tube (Terumo, Tokyo, Japan). After blood collection, blood was transferred into a 0.5-mL tube, and then subjected to centrifugation at 4°C and 3,400 G for 15 minutes. Subsequently, the supernatant was collected, and then cryopreserved at -80°C. A quality control serum, Consera (registered trademark) (Nissui pharmaceutical, Tokyo, Japan) was dissolved in 3 mL of distilled water (dH 2 O) per bottle.
  • dH 2 O distilled water
  • Consera 190 ⁇ L was mixed with the collected blood plasma and 10 ⁇ L of urine, thereby preparing samples.
  • PEG11(E)-TC11 and HOEtO-TC11 were each diluted with dH 2 O in such a manner that the concentrations were 0, 2, 4, 6, 8, and 10 ⁇ M, and then 10 ⁇ L of the solution at each concentration and 190 ⁇ L of Consera were mixed, thereby preparing a calibration curve sample.
  • Sep-Pak (registered trademark) C18 solid phase extraction cartridge (Waters Associates, Massachusetts, USA) was washed with 2 mL of 100% CH 3 CN, and then equilibrated with 4 mL of dH 2 O. Each sample and a sample for preparing a calibration curve were added to the cartridge, and then rinsed with 200 ⁇ L of dH 2 O. Subsequently, the resultant was washed with 2 mL of dH 2 O, 2 mL of 40%CH 3 CN, and 250 ⁇ L of 100% CH 3 CN, and then eluted with 1 mL of 100% CH 3 CN.
  • the eluate was evaporated under vacuum using a centrifugal evaporator CVE-2000 (TOKYO RIKAKIKAI CO, LTD, Tokyo, Japan), redissolution was performed with 100 ⁇ L of 100% EtOH, and then filtration was performed using a chromatodisc having a pore size of 0.45 ⁇ m (GL sciences, Tokyo, Japan).
  • HPLC High Performance Liquid Chromatography
  • the high performance liquid chromatography (High Performance Liquid Chromatography; HPLC) apparatuses used herein are the following apparatuses manufactured by JASCO (Tokyo, Japan). Intelligent HPLC pump PU-2089 Autosampler AS-2057 Column oven Co-2060 UV detector UV-2075 Fluorescence detector FP-2020 Chromatography data station LC-NetII/ADC
  • Inertsil registered trademark
  • PEG11(E)-TC11 was examined for stability in mouse blood plasma (A) and in medium (B). As shown in Fig. 3A , it was revealed that PEG11(E)-TC11 is very quickly hydrolyzed by esterase in mouse blood plasma, and converted to HOEtO-TC11. Moreover, as shown in Fig. 3B , PEG11(E)-TC11 was slowly degraded even in medium and then converted to HOEtO-TC11. Such degradation is considered to result from a small amount of esterase in FBS (fetal calf serum) contained in the medium.
  • FBS fetal calf serum
  • MM cell lines For examination of cell proliferation inhibitory activity, high-risk MM cell lines (KMM1, KMS11, KMS21, KMS26, KMS28, KMS34) established by Takemi Otsuki et al., of Kawasaki Medical School were used ( Otsuki, T. et al., Int. J. Oncol., 15: 1205-1212, 1999 ). MM cell line MUM24 was established by one of the present inventors, Yutaka Hattori by himself from patients resistant to thalidomide therapy ( Hattori, Y. et al., Blood Cancer J., 3: e115, 2013 ). The genetic abnormality of these cell types, and in particular deletion of p53 gene were examined by FISH method ( Chang, H.
  • KMM1, KMS11, KMS26, KMS28, KMS34, and MUM24 were found to have one p53 gene-deficient allele, and KMS21 was found to have alleles with no p53 deletion.
  • These high-risk MM cell lines were treated with drugs such as TC11, HOEtO-TC11, PEG11(E)-TC11, and PEG11(O)-TC11 at arbitrary concentrations. Cell viability after 48 hours was determined using an MTT assay method ( Mosman, T. J. Immunol. Methods, 65, 55-63, 1983 ), thereby calculating IC 50 values.
  • each high-risk MM cell line was prepared at a concentration of 2 ⁇ 10 5 cells/ml, and then seeded in an amount of 50 ⁇ l (1 ⁇ 10 4 cells) per well.
  • drugs such as TC11, HOEtO-TC11, PEG11(E)-TC11, and PEG11(O)-TC11 having arbitrary concentrations were added.
  • 10 ⁇ L each of an MTT labeling reagent in Cell Proliferation Kit I [MTT] (Roche Diagnostics, Basel, Switzerland) was added. After 4 hours of treatment at 37°C, 100 ⁇ L each of solution buffer was added and then left to stand overnight at 37°C.
  • drugs such as TC11, HOEtO-TC11, PEG11(E)-TC11, and PEG11(O)-TC11 exhibited significant cell proliferation inhibitory activity against many high-risk MM cell lines.
  • PEG11(E)-TC11 had a high drug effect, and its IC 50 value ranged from 1 to 7 ⁇ M, the level of which was almost the same as TC11's IC 50 value of 2-8 ⁇ M.
  • PEG11(O)-TC11 also exhibited significant cell proliferation inhibitory activity, but the drug effect (IC 50 value of 5-60 ⁇ M) varied for different cell lines.
  • PEG11(0)-TC11 had an IC 50 value 5 to 10 times higher and a drug effect lower than those of PEG11(E)-TC11.
  • HOEtO-TC11 had also a high drug effect (IC 50 value of 1-8 ⁇ M), the level of which was almost the same as that of TC11 or PEG11(E)-TC11. It was inferred from these results as follows.
  • PEG11(E)-TC11 is hydrolyzed by esterase in medium and within cells for conversion thereof to PEG11(E)-TC11 having a small molecular size, and then finally starts to act.
  • PEG11(E)-TC11 has high reactivity with a target molecule in view of molecular mobility and thus has a high drug effect.
  • high-risk MM cell lines were treated with drugs, TC11 and PEG5(E)-TC11 at arbitrary concentrations.
  • Cell viability after 48 hours was determined using a WST-1 assay method ( Cook, JA. & Mitchell, JB., Anal. Biochem., 179, 1-7, 1984 ), and thus IC 50 values were calculated.
  • each high-risk MM cell line was prepared at 5 ⁇ 10 5 cells/ml, and then seeded at 50 ⁇ l per well (2.5 ⁇ 10 4 cells).
  • Drugs such as TC11, and PEG5(E)-TC11 were added to wells at arbitrary concentrations.
  • WST-1 reagent was added at 48 hours after addition of drugs.
  • drugs such as TC11 and PEG5(E)-TC11 exhibited significant cell proliferation inhibitory activity against various high-risk MM cell lines, and the drug effect (IC 50 value of 1.3-4.6 ⁇ M) of PEGS(E)-TC11 was almost the same as the IC 50 value of TC11.
  • MM cell lines (KMM1, KMS11, KMS21, KMS26, KMS27, KMS28, KMS34) used herein were those established by Takemi Otsuki et al., of Kawasaki Medical School ( Otsuki, T. et al., Int. J. Oncol., 15: 1205-1212, 1999 ). MUM24 used herein was established by one of the present inventors, Yutaka Hattori by himself from patients resistant to thalidomide therapy ( Hattori, Y. et al., Blood Cancer J., 3: e115, 2013 ).
  • PEG11(E)-TC11 exhibited significant in vitro cell proliferation inhibitory activity against various high-risk MM cells, however, lenalidomide did not exhibit in vitro cell proliferation inhibitory activity, against myeloma cells other than KMS21 and MUM24, including high-risk MM cells, KMS11, KMS28, and KMS34, as shown in Fig. 5B .
  • Cancer cells used herein were KMS34 (human myeloma cells), HeLa (human cervical cancer cells), HCT116 (human colon cancer cells; p53+/+), KATOIII (human stomach cancer cells), MDA-MB-231 (human breast cancer cells), HT-29 (human colon adenocarcinoma cells), PANC-1 (human pancreatic cancer cells), and Li-7 (human liver cancer cells).
  • Proliferation assay of various cancer cells was performed by the WST-1 method ( Cook, JA. & Mitchell, JB., Anal. Biochem., 179, 1-7, 1984 ) as follows.
  • PEG11(E)-TC11 and PEG11(O)-TC11 exhibit in vitro cell proliferation inhibitory activity against various cancer cells. It could be confirmed that PEG11(E)-TC11 exhibits a significant drug effect (IC 50 value of 0.67 ⁇ M) on cancer cells and particularly KMS34 (myeloma cells), and also exhibits strong cell proliferation inhibitory activity against HeLa (human cervical cancer cells), KATOIII (human stomach cancer cells), and HCT116 (human colon cancer cells).
  • Proliferation assay of cancer cells and normal cells was performed using WST-1 as follows. One ⁇ l each of PEG11(E)-TC11 and TC11 prepared at target concentrations was added to each well of a 96-well microplate. Medium was added to the wells at 50 ⁇ l per well, and then mixed by tapping lightly the plate. Next, high-risk MM cells (KMS34) prepared at 5 ⁇ 10 5 cells/ml and bone marrow stromal cell-derived normal cells (WT) prepared at 2 ⁇ 10 5 cells/ml were seeded at 50 ⁇ l (2.5 ⁇ 10 4 cells and 1 ⁇ 10 4 cells, respectively) per well, and then incubated in a CO 2 incubator (37°C, 5% CO 2 ) for 48 hours.
  • KMS34 high-risk MM cells
  • WT bone marrow stromal cell-derived normal cells
  • PEG11(E)-TC11 and TC11 each exhibited significant proliferation inhibitory activity against high-risk MM cells (KMS34), but exhibited no proliferation inhibitory activity against bone marrow stromal cell-derived normal cells (WT). These results successfully confirmed that PEG11(E)-TC11 is a drug exhibiting cell proliferation inhibitory activity in a cancer-specific manner against various cancer cells including MM cells.
  • MUM24 cells were treated with TC11 and PEG11(E)-TC11, Annexin V/PI staining was performed, and then analysis was conducted using a flow cytometer. More specifically, MUM24 was adjusted at 2.0 ⁇ 10 5 cells/mL, and then 1.95 mL each thereof was seeded to a 6-well plate. From the next day, TC11 and PEG 11 (E)-TC11 were added in such a manner that the final concentrations were 0, and 10 ⁇ M, and then cells were cultured at 37°C for 24, 48, and 72 hours.
  • each cell (1.5 ml each) was collected, and then washed twice with Phosphate Buffered Saline (PBS, Sigma-Aldrich). Subsequently, each resultant was suspended again in a solution prepared by adding 5 ⁇ L of Annexin V-FITC and 5 ⁇ L of Propidium Iodide (PI) to 500 ⁇ L of 1 ⁇ Binding Buffer in Annexin V-FITC Apoptosis Detection Kit (Bio Vision, California, USA), and then left to stand in the dark for 5 minutes. Fluorescence intensity was measured using BDTM LSR II Flow Cytometer (Becton, Dickinson and company, New Jersey, USA). Wavelengths to be measured were excitation wavelength of 488 nm, and fluorescence wavelengths of 530 ⁇ 15 nm for Annexin V-FITC and 610 ⁇ 10 nm for PI.
  • both TC11 and PEG11(E)-TC11 were confirmed to cause an increase in, an early apoptosis fraction, Annexin V + /PI - cells, and an increase in, a late apoptosis fraction, Annexin V + /PI + cells.
  • TC11 and PEG11(E)-TC11 clearly induce apoptosis in MM cells.
  • the present inventors have reported that when HeLa cells of a human cervical cancer-derived cell line are treated with TC11, multipolarization and then multinucleation take place ( Shiheido, H. et al., PLoS One, 7: e38878, 2012 ). The present inventors have also reported in the same paper ⁇ -tubulin and NPM1 as binding molecules for TC11. Next, intracellular DNA content was measured using flow cytometry in order to examine the effects of TC11 and its derivative, PEG11(E)-TC11 on cell cycle. More specifically, MUM24 was adjusted at 4.0 ⁇ 10 5 cells/mL, 1.95 mL each of MUM24 was seeded to a 6-well plate.
  • TC11 and PEG11(E)-TC11 were added in such a manner that the final concentrations were 0 and 3 ⁇ M, respectively, and then cells were cultured at 37°C for 12, 24, and 36 hours. Each cell was collected, and then washed with PBS. 70% EtOH (460 ml) was added and then the resultant was left to stand overnight at - 30°C. After washing with PBS, 100 ml of citrate-phosphate buffer was added, and then the resultant was left to stand at room temperature for 30 minutes, thereby eluting fragmented DNA.
  • MUM24 cells treated with TC11 and the same treated with PEG11(E)-TC11 were each found to be composed of a higher proportion of tetraploid cells than that of the control. Therefore, it was successfully confirmed that TC11 and PEG 11 (E)-TC11 induce cell death by causing G2/M phase arrest in high-risk MM cells (MUM24).
  • Protein cereblon (cereblon; CRBN) binding as a teratogen to thalidomide has been recently identified ( Ito, T. et al., Science, 327: 1345-1350, 2010 ).
  • thalidomide was confirmed to bind to CRBN, with its glutarimide ring part ( Chamberlain, PP, et al., Nature Struct. Mol. Biol., 21: 803-810, 2014 ).
  • TC11 lacks a glutarimide ring serving as a CRBN binding site of thalidomide, lenalidomide or the like, but instead has a benzene ring having a bulky diisopropylphenyl group as a result of substitution, and thus is inferred to bind to CRBN with difficulty.
  • TC11 lacks a glutarimide ring serving as a CRBN binding site of thalidomide, lenalidomide or the like, but instead has a benzene ring having a bulky diisopropylphenyl group as a result of substitution, and thus is inferred to bind to CRBN with difficulty.
  • TC11 lacks a glutarimide ring serving as a CRBN binding site of thalidomide, lenalidomide or the like, but instead has a benzene ring having a bulky diisopropylphenyl group as a result of substitution, and thus is infer
  • CRBN was prepared as follows. A T7 tag was added 5'-upstream of, and a FLAG tag and a histidine tag were added 3'-downstream of a full-length CRBN gene. The gene was introduced into a pcDNA3.3 expression vector (Invitrogen), thereby constructing a plasmid DNA. Next, a Kozak sequence was added 5'-upstream of the T7 tag using a KOD-Plus-Mutagenesis Kit (TOYOBO). The plasmid DNA was transfected into 293T cells.
  • Protein bands corresponding to CRBN were separated by SDS-PAGE, and then detected by Western blot. As shown in Fig. 9A , it was revealed that CRBN bound to thalidomide immobilized on beads, but did not bind to TC11 immobilized on beads. Note that "I” indicates "Input fraction”.
  • CRBN having the histidine-tagged C terminus prepared by the above method was used as a ligand (substance to be adhered to a sensor chip) of a BIACORE biosensor.
  • CRBN was immobilized on a Sensor Chip NTA.
  • TBS Nacalai Tesque
  • CRBN (10 ⁇ l) was injected after buffer exchange for immobilization of CRBN to flow cell 2 and flow cell 4, and thus 7800 RU (response unit) of CRBN bound to each of them.
  • TC11 and its derivatives, PEG11(E)-TC11, PEG11(O)-TC11, and HOEtO-TC11 and thalidomide or lenalidomide were dissolved as analytes (substances to be applied to a flow system) in TBS (Nacalai Tesque) buffers, and the concentrations were adjusted at 1 ⁇ M, 2 ⁇ M, and 10 ⁇ M.
  • Analysis of intermolecular interaction using BIACORE 3000 was conducted using TBS buffer at a flow rate of 20 ⁇ l/min. These analytes were injected, 60 ⁇ l each, using FLOWPATH 1,2 or FLOWPATH 3,4 and KINJECT, and then dissociation was measured for 300 seconds.
  • TC11 and its derivatives PEG11(E)-TC11, PEG11(O)-TC11, and HOEtO-TC11, and thalidomide or lenalidomide were measured using TBS buffers at concentrations of 1 ⁇ M, 2 ⁇ M, and 10 ⁇ M. Analysis was conducted as follows. A value obtained by subtracting the sensorgram of flow cell 1 from the sensorgram of flow cell 2, or, a value obtained by subtracting the sensorgram of flow cell 3 from the sensorgram of flow cell 4 was analyzed by reaction kinetic analysis using analysis software BIA evaluation ver. 4.1 and 1:1 binding model, thereby determining the dissociation constant (K D ).
  • Fig. 9B shows the surface plasmon resonance biosensorgram of the interaction of CRBN with thalidomide and lenalidomide. Gray denotes actual measured data, and black denotes fitting curves. Good fitting curves were obtained for both in this measurement.
  • Thalidomide exhibited a K D value of 5.66x 10 -7 M for CRBN.
  • lenalidomide exhibited a K D value of 3.65 ⁇ 10 -6 M for CRBN.
  • Fig. 9C shows the surface plasmon resonance biosensorgram of the interaction of CRBN with TC11 and its derivatives, HOEtO-TC11, PEG11(E)-TC11, and PEG11(O)-TC11.
  • TC11 and PEG11(E)-TC11 Mouse blood kinetics upon single intraperitoneal administration of TC11 and PEG11(E)-TC11 was examined.
  • TC11 (60 mg/kg BW) and PEG11(E)-TC11 (177.3 mg/kg BW) in an equimolar amount thereof were administered to six 5-week-old male ICR mice (CLEA Japan, Tokyo, Japan) via single intraperitoneal administration.
  • TC11 (60 mg/kg BW) and PEG11(E)-TC11 (177.3 mg/kg BW) in an equimolar amount thereof were administered to six 5-week-old male ICR mice (CLEA Japan, Tokyo, Japan) via single intraperitoneal administration.
  • a heparinized hematocrit capillary tube Terumo, Tokyo, Japan.
  • Example 7 After blood collection, centrifugation was performed at 4°C and 3,400 G for 15 minutes, 10 ⁇ l of a supernatant was collected, and then cryopreserved at -80°C.
  • the subsequent serum treatment and HPLC analyses of TC11 and PEG11(E)-TC11 and HOEtO-TC11 were performed by the same method as in Example 7. Note that as described in Example 7, since PEG11(E)-TC11 is quickly hydrolyzed by esterase in blood and then converted to HOEtO-TC11, the blood HOEtO-TC11 level was measured.
  • tmax the time at which the blood level reached the highest level (tmax), the highest blood level (Cmax), and the serum half-life (t1/2) upon single intraperitoneal administration of TC11 (60 mg/kg BW) were 1.5 hours, 2.6 ⁇ M, and 1.4 hours, respectively.
  • tmax, Cmax, and t1/2 upon single intraperitoneal administration of PEG11(E)-TC11 (177.3 mg/kg BW) were 1.0 hour, 24.4 ⁇ M, and 2.2 hours, respectively, confirming the highest blood level 9 times higher than that of TC11 and the prolonged serum half-life 1.6 times longer than that of TC11.
  • TC11 and its derivatives were examined for in vivo antitumor effects using 6 KMS11 xenograft model mice.
  • KMS11 was administered to 6-week-old ICR/SCID mice at 3 ⁇ 10 7 cells/mouse via hypodermic injection. Tumor volume was measured over time. The day on which each tumor volume exceeded 50 mm 3 was designated as Day 1 and observation was performed for 14 days.
  • the single dosage of TC11 was 60 mg/kgBW, the intraperitoneal dosages of PEG11(E)-TC11 and PEG11(O)-TC11 were 177.3 mg/kgBW and 163.8 mg/kgBW, respectively, which were equimolar amounts of that of TC11.
  • TC11 is hardly dissolved in physiological saline (NaCl 0.9 w/v%) alone, TC11 was dissolved in DMSO, and then diluted with 1.1% Tween 80/physiological saline, so that it had a predetermined concentration. The final composition of the solvent was 10% DMSO + 1% Tween 80/physiological saline. TC11 was almost completely dissolved under the solvent conditions. PEG11(E)-TC11 and PEG11(O)-TC11 were each completely dissolved in physiological saline. Drug administration was performed for two out of every 3 days (Day 1, 2, 4, 5, 7, 8, 10, 11, 12, 13). Mice were sacrificed on Day 14, and then tumor weights were measured and in vivo findings were confirmed.
  • HOEtO-TC11 had a drug effect higher than that of TC11 in vivo, and whether or not lenalidomide exhibited an antitumor effect in vivo on high-risk MM cells KMS11 were examined.
  • TC11, HOEtO-TC11, and lenalidomide were examined for the antitumor effect in vivo using 5 KMS11 xenograft model mice.
  • KMS11 was administered to 5-week-old ICR/SCID mice at 2 ⁇ 10 7 cells/mouse via hypodermic injection. Tumor volumes were measured over time, and the day on which the tumor volume exceeded 50 mm 3 was designated as Day 1 and observation was performed for 9 days.
  • Single dosage of TC11 was 60 mg/kgBW, the intraperitoneal dosages of HOEtO-TC11 and lenalidomide were 71.1 mg/kg BW and 49.9 mg/kgBW, respectively, which were equimolar amounts of TC11.
  • KMS-11 tumor growth was suppressed by administration of TC11 and HOEtO-TC11 to a degree higher than that in control individual mice to which no drug had been administered, resulting in a clear significant difference.
  • administration of HOEtO-TC11 significantly inhibited KMS-11 tumor growth to a level about 1/3 of that of the control, and about a half of that of TC11.
  • the symbol "*" in the figure indicates that the t-test result (significance level 5%) was more significant than that of one control group.
  • lenalidomide had an in vivo anti-tumor effect clearly weaker than that of HOEtO-TC11.
  • Pd/C (K-type) (2.3 g) was added to a solution (mixture of 200 mL of EtOH and 200 mL of THF) of 7.0 g of hydroxyethoxy-la (16.97 mmol), and then the mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours.

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